Despite convincing evidence for a heritable component in alcohol use disorders (AUD) and other drug addictions (DA), robust and reproducible evidence for genes involved in AUD or DA is rare. One of the few consistent findings over the past 10 years are genetic associations of addiction and related endophenotypes such as brain oscillations and impulsivity with SNPs in GABRA2, the gene encoding the GABA(A) alpha 2 receptor. The biological basis of this association has, however, remained elusive. Associated SNPs are in strong linkage disequilibrium (LD) with each other. Most Caucasians carry one or two common haplotypes differing at 299 SNP alleles, which do not affect an amino acid or measured overall mRNA levels. Since GABRA2 is only expressed in brain, functional studies have so far been difficult. Here, we hypothesize that SNPs within GABRA2 affect gene regulation and/or splicing. Computational integration of chromatin data across 30+ cell types indicates the presence of brain-specific enhancers within the GABRA2 gene, with several SNPs of the relevant haplotype within predicted regulatory regions. Preliminary RNASeq and Sanger Sequencing data suggest allelic imbalance, with the non-ancestral, risk increasing haplotype associated with increased transcription. In Aim 1, we will test the enhancer capability of 8 DNA fragments with either of the two SNP alleles in- vitro in existing human embryonic stem cells differentiated into neurons using standard reporter assays. In Aim 2, to investigate the effect of GABRA2 haplotypes on gene regulation, we will generate neurons that differ by GABRA2 haplotype by generating induced pluripotent stem cell lines (iPSCs) from lymphoblastoid cell lines (LCLs) from four sets of siblings, hence in a similar genetic background. These cell lines will allow, in a genetically similar and well controlled background, and throughout the differentiation from iPSC to neurons, the study of a) transcription level as allelic imbalance, b) GABRA2 splicing at several differentially spliced site, and c) changes of methylation of CpG sites within GABRA2 during differentiation. Our studies will elucidate the path from genetic variation to addiction. Our studies will generate iPSC lines from related individuals who differ by GABRA2 haplotype, which will allow study of development of GABRA2 gene expression over differentiation to neurons. These cell lines will also be made available to others, as they may be useful for the study of other common neuron-specific variants. Future studies building on this research and the tools generated by this R21 will include genetic fine mapping of the molecular phenotype in iPSCs of recombinant cell lines, electrophysiology, reactivity of neurons to GABA, and response to electrical stimulation, i.e. the cellular and physiological phenomena that go beyond proximal RNA and protein, on the path to brain circuitry.